The present invention relates to a novel cytokine that is important in host defense and immunity against infection and for the processes for obtaining the purified factor by recombinant genetic engineering techniques.
A family of regulatory proteins that deliver signals between many different types of cells in the body has been identified. These regulatory molecules are known as cytokines. Many of the cytokines have been found to control the growth and development and biological activities of cells in the hematopoietic and immune systems. Cytokines have also been identified which are produced by other cell types including fibroblasts and endothelial cells which transmit signals between these cells and a variety of responsive target cells. This family of cytokines is clearly important for maintaining homeostasis and for coordinating the physiological responses to a variety of insults including wounding and infection as well as regulating the immune response [See, for example G. Wong and S. Clark, Immunology Today, 9(5):139 (1988)]. The family of cytokines includes the interleukins, the hematopoietic colony-stimulating factors, the interferons, and the tumor necrosis factors among others. In addition, two subfamilies within the larger cytokine family have emerged that share evolutionary relatedness at the nucleotide level. Members of one of these families share sequence similarity with a cytokine known as macrophage inflammatory protein 1 (MIP-1) [Davatelis, G. et al J. Exp. Med., 167:1939-1944 (1988)], while members of the other family share sequence similarity with a second macrophage inflammatory protein, MIP-2 [Wolpe, S. D. et al, Proc. Nat""l Acad. Sci. USA, 86:612-616 (1988)]. MIP-1 and MIP-2 are cytokines produced by activated macrophages that induce local inflammatory responses when injected subcutaneously in mice. Other polypeptides have been identified through molecular biological approaches which are clearly related to either MIP-1 or MIP-2 but for which biological activities have not yet been identified. Although the function of these molecules is not known, they, like other members of the cytokine family, are likely to be important in various aspects of regulating homeostasis or coordinating physiological responses to wounding, injury, or infection or in the regulation of the immune system.
One member of the MIP-1 subfamily may be the murine JE [Rollins et al, Proc. Nat.""l. Acad. Sci. USA 85:3738-3752 (1988)] and its human homolgue disclosed herein.
In one aspect the present invention provides, substantially free from co-produced polypeptides, a novel human cytokine herein termed JE which is elicited in response to platelet-derived growth factor (PDGF). JE may be characterized by containing the predicted amino acid sequence from at least amino acid #30 to #99 as set forth in Table I. This novel factor when expressed in COS cells displays considerable size heterogeneity with three predominant species present with estimated sizes of approximately 15,500, 15,000, and 13,000 as determined by SDS-PAGE. Additional microheterogeneous species are present with molecular weights from 16,000-18,000 daltons.
In one aspect, the invention provides JE factor produced by culturing a cell transformed with the DNA sequence comprising the sequence of Table I from at least nucleotide #73 to #772 and recovering and purifying from the culture medium a protein comprising the amino acid sequence from amino acid #30 to #99 of Table I.
Another aspect of the invention includes DNA sequences coding on expression for a human JE polypeptide. One such DNA sequence is the same or substantially the same as the approximately 772 nucleotide sequence which appears below in Table I.
Also provided by the present invention are vectors containing a DNA sequence encoding JE in operative association with an expression control sequence. Host cells transformed with such vectors for use in producing recombinant JE are also provided by the present invention.
The vectors and transformed cells of the invention are employed in another aspect, a novel process for producing recombinant human JE polypeptide. In this process a cell line transformed with a DNA sequence encoding JE polypeptide in operative association with an expression control sequence therefor is cultured. This claimed process may employ a number of known cells as host cells for expression of the polypeptide. Presently preferred cell lines are mammalian cell lines, and bacterial cells.
Another aspect of this invention provides pharmaceutical compositions comprising a therapeutically effective amount of JE in a pharmaceutically acceptable vehicle. Because JE expression is activated by PDGF, a growth factor released by platelets at the site of a wound, JE protein is likely to be useful directly for treating wounds. JE is also likely to have other cytokine properties including the ability to enhance host defense or to stimulate the hematopoietic or immune systems. Therefore, the pharmaceutical compositions of the invention may be useful in the treatment of cancer or in potentiating the efficacy of vaccines. Generally, it is contemplated that compositions of the invention may be useful for the treatment of disease states which involve immune system deficiencies.
A further aspect of the invention, therefore, is a method for treating tissue injuries or accelerating wound healing by administering to a patient a therapeutically effective amount of JE in a suitable pharmaceutical carrier. Further included are methods for treating cancer, diseases characterized by a deficiency in the number or level of activity of hematopoietic cells, or potentiating the efficacy of vaccines by administering to a patient a therapeutically effective amount of JE in a suitable pharmaceutical carrier. These therapeutic methods may include administering simultaneously or sequentially with JE polypeptides an effective amount of at least one other cytokine, hematopoietin, interleukin, growth factor, or antibody.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.